Cardiomyocytes are thought to be terminally differentiated. Although a small percentage of the cells may have proliferative capacity, it is not sufficient to replace injured or dead cardiomyocytes. Death of cardiomyocytes occurs, for example, when a coronary vessel is occluded by a thrombus and the surrounding cardiomyocytes cannot be supplied with necessary energy sources from other coronary vessels. Loss of functional cardiomyocytes may lead to chronic heart failure.
The proliferative capacity of the cardiomyocytes is not sufficient to regenerate the heart following myocardial injury. Conventional pharmacological therapy for patients with different stages of ischemic heart disease improves cardiac function, survival and quality of life. However, ischemic heart disease is still the most life-threatening disease in western society and alternative therapies will be necessary to improve the clinical outcome for patients with ischemic heart disease further. In recent years, the focus on cell replacement therapy has been intensified, stimulated by the increasing number of potential cell sources for transplantation, such as skeletal myoblasts, adult cardiac stem cells, bone marrow stem cells and embryonic stem cells.
A potential route for restoring “normal” heart function is replacement of injured or dead cardiomyocytes by new functional cardiomyocytes. Human embryonic stem (hES) cells are a potential source of cells for cardiomyocyte replacement. Either spontaneously, or upon induction, differentiation of hES cells into cardiomyocytes can be achieved.
Embryonic stem cells can differentiate to cardiomyocytes in culture, opening the possibility for controlled in vitro studies of developing human heart cells. However, it is difficult to control and induce the specific differentiation of ES cells solely towards cardiomyocytes (Brand, 2003).
Co-culturing END2 cells with hES cells can induce cardiomyocyte differentiation of the hES cells in serum-free culture conditions (Passier, et al, 2005). An END2 conditioned media (END2-CM) system has also been demonstrated to induce robust differentiation of cardiomyocytes in hES-derived embryoid bodies. Using feeder-free conditioned media allows for a more clean and controlled differentiation of cardiomyocytes than co-culturing. However, the END2-CM contains proteins and other molecules released from mouse END2 cells and as such hES cell-derived cardiomyocytes cultured from END2-CM would be considered a “xenoproduct” for clinical purposes. Therefore, identification of the “factor(s)” produced by the END2 cell line is of paramount importance to the development of a hES-derived therapeutic product.
Identifying the END2 inducing factors and other factors involved in the differentiation process has been challenging and illusive since the generation of the visceral-endoderm-like END2 cell line from a mouse P19 embryonic carcinoma cell line (Mummery, 1991). Earlier data has suggested that the END2 “factor” is secreted and it is a protein (van den et al, 1991). Regardless of its uncertainty on whether it is protein, it is important to identify the factor(s) if a therapeutically acceptable product is to be developed. Identifying cardiomyocyte inducing factors will provide opportunities to develop clinically compliant populations of hES cell-derived cardiomyocytes.
If differentiation conditions can be established with defined culturing conditions, and without the potential presence of animal pathogens, hES cell derived cardiomyocytes may be produced safely which are suitable for cardiomyocyte transplantation in patients with heart disease.
Accordingly, the invention seeks to identify factors that are involved in the process of cardiomyocyte differentiation and provide a defined culture system that is suitable for the induction of stem cells to cardiomyocytes and cardiac progenitors.